Microtubules (MTs) are essential dynamic polymers required for chromosome segregation and intracellular organization, and are the direct targets of anti-cancer chemotherapeutics like taxol and the Vinca alkaloids. The dynamic properties of MTs are central to their function, and they derive from the structural and biochemical properties of individual tubulin subunits and how they interact within the MT lattice. It is increasingly appreciated that tubulin subunits adopt distinct conformations as part of the GTPase-dependent polymerization dynamics, and that regulatory proteins selectively recognize subsets of these conformations to control MT elongation, stability, and switching. The long-term goal of this research is to build a structural understanding of how allostery and the tubulin conformation cycle dictate MT dynamics, and of the mechanisms by which regulatory factors control MT dynamics. In prior project periods, we pioneered a powerful approach based on structure-inspired site-directed ??-tubulin mutants. In the present proposal, through three specific aims, we will build on these themes to provide unique and fundamental new insights into the physical origins and regulatory mechanism of MT dynamics. We will use biochemistry, reconstitution, and modeling to define general biochemical mechanisms for XMAP215-family polymerase activity and processivity. We will reveal through structures how an `allosteric' mutation that alters MT dynamics affects tubulin conformation in human and yeast MTs, and we will provide new conformation cycle mutants to expand our understanding of allostery in MT dynamics. Finally, we will identify biochemical and structural design principles underlying how CLASP TOG interactions with tubulin suppress catastrophe and promote rescue. This work will provide new information about the conformation(s) of ??-tubulin and how `allosteric' mutations can perturb MT dynamics and tubulin conformation. The work will also expand our understanding of how different TOG domains achieve different regulatory outcomes, with implications for the underlying mechanisms of microtubule dynamics.